Methods and interferon deficient substrates for the propagation of viruses

ABSTRACT

The present invention relates, to novel methods and substrates for the propagation of viruses. The invention relates to IFN-deficient substrates and methods for propagating viruses in these unconventional substrates. In particular, the invention relates to methods of propagating viruses in immature embryonated eggs, preferably six- to nine-day-old chicken eggs. The methods of the invention are particularly attractive for growing viruses suitable for use in vaccine and pharmaceutical formulations.

This application is a continuation-in-part of application Ser. No.60/117,683 filed Jan. 29, 1999; application Ser. No. 60/108,832 filedNov. 18, 1998; and application Ser. No. 60/089,103 filed Jun. 12, 1998,each of which is incorporated by reference in its entirety herein.

The work reflected in this application was supported, in part, by agrant from the National Institutes of Health, and the Government mayhave certain rights to the invention.

1. INTRODUCTION

The present invention relates, in general, to attenuated negative-strandRNA viruses having an impaired ability to antagonize the cellularinterferon (IFN) response, and the use of such attenuated viruses invaccine and pharmaceutical formulations. The invention also relates tothe development and use of IFN-deficient systems for the selection,identification and propagation of such attenuated viruses.

In a particular embodiment, the invention relates to attenuatedinfluenza viruses having modifications to the NS1 gene that diminish oreliminate the ability of the NS1 gene product to antagonize the cellularIFN response. The mutant viruses replicate in vivo, but demonstratereduced pathogenicity, and therefore are well suited for use in livevirus vaccines, and pharmaceutical formulations.

2. BACKGROUND OF THE INVENTION 2.1 The Influenza Virus

Virus families containing enveloped single-stranded RNA of thenegative-sense genome are classified into groups having non-segmentedgenomes (Paramyxoviridae, Rhabdoviridae, Filoviridae and Borna DiseaseVirus) or those having segmented genomes (Orthomyxoviridae, Bunyaviridaeand Arenaviridae). The Orthomyxoviridae family, described in detailbelow, and used in the examples herein, includes the viruses ofinfluenza, types A, B and C viruses, as well as Thogoto and Dhoriviruses and infectious salmon anemia virus.

The influenza virions consist of an internal ribonucleoprotein core (ahelical nucleocapsid) containing the single-stranded RNA genome, and anouter lipoprotein envelope lined inside by a matrix protein (M1). Thesegmented genome of influenza A virus consists of eight molecules (sevenfor influenza C) of linear, negative polarity, single-stranded RNAswhich encode ten polypeptides, including: the RNA-dependent RNApolymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which formthe nucleocapsid; the matrix membrane proteins (M1, M2); two surfaceglycoproteins which project from the lipid containing envelope:hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein(NS1) and nuclear export protein (NEP). Transcription and replication ofthe genome takes place in the nucleus and assembly occurs via budding onthe plasma membrane. The viruses can reassort genes during mixedinfections.

Influenza virus adsorbs via HA to sialyloligosaccharides in cellmembrane glycoproteins and glycolipids. Following endocytosis of thevirion, a conformational change in the HA molecule occurs within thecellular endosome which facilitates membrane fusion, thus triggeringuncoating. The nucleocapsid migrates to the nucleus where viral mRNA istranscribed. Viral mRNA is transcribed by a unique mechanism in whichviral endonuclease cleaves the capped 5′-terminus from cellularheterologous mRNAs which then serve as primers for transcription ofviral RNA templates by the viral transcriptase. Transcripts terminate atsites 15 to 22 bases from the ends of their templates, where oligo(U)sequences act as signals for the addition of poly(A) tracts. Of theeight viral RNA molecules so produced, six are monocistronic messagesthat are translated directly into the proteins representing HA, NA, NPand the viral polymerase proteins, PB2, PB1 and PA. The other twotranscripts undergo splicing, each yielding two mRNAs which aretranslated in different reading frames to produce M1, M2, NS1 and NEP.In other words, the eight viral RNA segments code for ten proteins: ninestructural and one nonstructural. A summary of the genes of theinfluenza virus and their protein products is shown in Table I below.

TABLE I INFLUENZA VIRUS GENOME RNA SEGMENTS AND CODING ASSIGNMENTS^(a)Length_(b) Encoded Length_(d) Molecules Segment (Nucleotides)Polypeptide_(c) (Amino Acids) Per Virion Comments 1 2341 PB2 759 30-60RNA transcriptase component; host cell RNA cap binding 2 2341 PB1 75730-60 RNA transcriptase component; initiation of transcription 3 2233 PA716 30-60 RNA transcriptase component 4 1778 HA 566 500 Hemagglutinin;trimer; envelope glycoprotein; mediates attachment to cells 5 1565 NP498 1000 Nucleoprotein; associated with RNA; structural component of RNAtranscriptase 6 1413 NA 454 100 Neuraminidase; tetramer; envelopeglycoprotein 7 1027 M₁ 252 3000 Matrix protein; lines inside of envelopeM₂ 96 ? Structural protein in plasma membrane; spliced mRNA 8 890 NS₁230 Nonstructural protein;function unknown NEP 121 ? Nuclear exportprotein; spliced mRNA ^(a)Adapted from R.A. Lamb and P. W. Choppin(1983), Annual Review of Biochemistry, Volume 52, 467-506. _(b)ForA/PR/8/34 strain _(c)Determined by biochemical and genetic approaches_(d)Determined by nucleotide sequence analysis and protein sequencing

The influenza A virus genome contains eight segments of single-strandedRNA of negative polarity, coding for one nonstructural and ninestructural proteins. The nonstructural protein NS1 is abundant ininfluenza virus infected cells, but has not been detected in virions.NS1 is a phosphoprotein found in the nucleus early during infection andalso in the cytoplasm at later times of the viral cycle (King et al.,1975, Virology 64: 378). Studies with temperature-sensitive (ts)influenza mutants carrying lesions in the NS gene suggested that the NS1protein is a transcriptional and post-transcriptional regulator ofmechanisms by which the virus is able to inhibit host cell geneexpression and to stimulate viral protein synthesis. Like many otherproteins that regulate post-transcriptional processes, the NS1 proteininteracts with specific RNA sequences and structures. The NS1 proteinhas been reported to bind to different RNA species including: vRNA,poly-A, U6 snRNA, 5′ untranslated region as of viral mRNAs and ds RNA(Qiu et al., 1995, RNA 1: 304; Qiu et al., 1994, J. Virol. 68: 2425;Hatada Fukuda 1992, J Gen Virol. 73:3325-9. Expression of the NS1protein from cDNA in transfected cells has been associated with severaleffects: inhibition of nucleo-cytoplasmic transport of mRNA, inhibitionof pre-mRNA splicing, inhibition of host mRNA polyadenylation andstimulation of translation of viral mRNA (Fortes, et al., 1994, EMBO J.13: 704; Enami, et al, 1994, J. Virol. 68: 1432; de la Luna, et al.,1995, J. Virol. 69:2427; Lu, et al., 1994, Genes Dev. 8:1817; Park, etal., 1995, J. Biol Chem. 270, 28433; Nemeroff et al., 1998, Mol. Cell.1:991; Chen, et al., 1994 EMBO J. 18:2273-83).

2.2 Attenuated Viruses

Inactivated virus vaccines are prepared by “killing” the viral pathogen,e.g., by heat or formalin treatment, so that it is not capable ofreplication. Inactivated vaccines have limited utility because they donot provide long lasting immunity and, therefore, afford limitedprotection. An alternative approach for producing virus vaccinesinvolves the use of attenuated live virus vaccines. Attenuated virusesare capable of replication but are not pathogenic, and, therefore,provide for longer lasting immunity and afford greater protection.However, the conventional methods for producing attenuated virusesinvolve the chance isolation of host range mutants, many of which aretemperature sensitive; e.g., the virus is passaged through unnaturalhosts, and progeny viruses which are immunogenic, yet not pathogenic,are selected.

A conventional substrate for isolating and growing influenza viruses forvaccine purposes are embryonated chicken eggs. Influenza viruses aretypically grown during 2-4 days at 37° C. in 10-11 day old eggs.Although most of the human primary isolates of influenza A and B virusesgrow better in the amniotic sac of the embryos, after 2 to 3 passagesthe viruses become adapted to grow in the cells of the allantoic cavity,which is accessible from the outside of the egg (Murphy, B. R., and R.G. Webster, 1996. Orthomyxoviruses p. 1397-1445. In Fields Virology.Lippincott-Raven P. A.)

Recombinant DNA technology and genetic engineering techniques, intheory, would afford a superior approach to producing an attenuatedvirus since specific mutations could be deliberately engineered into theviral genome. However, the genetic alterations required for attenuationof viruses are not known or predictable. In general, the attempts to userecombinant DNA technology to engineer viral vaccines have mostly beendirected to the production of subunit vaccines which contain only theprotein subunits of the pathogen involved in the immune response,expressed in recombinant viral vectors such as vaccinia virus orbaculovirus. More recently, recombinant DNA techniques have beenutilized in an attempt to produce herpes virus deletion mutants orpolioviruses which mimic attenuated viruses found in nature or knownhost range mutants. Until 1990, the negative strand RNA viruses were notamenable to site-specific manipulation at all, and thus could not begenetically engineered.

Attenuated live influenza viruses produced thus far might not be capableof suppressing the interferon response in the host in which theyreplicate. Therefore, although these viruses are beneficial because theyare immunogenic and not pathogenic, they are difficult to propagate inconventional substrates for the purposes of making vaccines.Furthermore, attenuated viruses may possess virulence characteristicsthat are so mild as to not allow the host to mount an immune responsesufficient to meet subsequent challenges.

3. SUMMARY OF THE INVENTION

The present invention relates to attenuated negative strand RNA viruseshaving an impaired ability to antagonize the cellular IFN response, andthe use of such viruses in vaccine and pharmaceutical formulations. Themutant viruses with an impaired IFN antagonist activity areattenuated—they are infectious, can replicate in vivo to providesubclinical levels of infection, and are not pathogenic. Therefore, theyare ideal candidates for live virus vaccines. Moreover, the attenuatedviruses can induce a robust IFN response which has other biologicalconsequences in vivo, affording protection against subsequent infectiousdiseases and/or inducing antitumor responses. Therefore, the attenuatedviruses can be used pharmaceutically, for the prevention or treatment ofother infectious diseases, cancer in high risk individuals, and/orIFN-treatable diseases.

The negative strand RNA viruses used in accordance with the inventioninclude both segmented and non-segmented viruses; preferred embodimentsinclude but are not limited to influenza virus, respiratory syncytialvirus (RSV), Newcastle disease virus (NDV), vesicular stomatitis virus(VSV), and parainfluenza virus (PIV). The viruses.used in the inventionmay be selected from naturally occurring strains, variants or mutants;mutagenized viruses (e.g., generated by exposure to mutagens, repeatedpassages and/or passage in non-permissive hosts); reassortants (in thecase of segmented viral genomes); and/or genetically engineered viruses(e.g. using the “reverse genetics” techniques) having the desiredphenotype—i.e., an impaired ability to antagonize the cellular IFNresponse. The mutant or genetically engineered virus can be selectedbased on differential growth in IFN deficient systems versus IFNcompetent systems. For example, viruses which grow in an IFN deficientsystem, but not in an IFN competent system (or which grow less well inan IFN competent system) can be selected.

The attenuated virus so selected can itself be used as the activeingredient in vaccine or pharmaceutical formulations. Alternatively, theattenuated virus can be used as the vector or “backbone” ofrecombinantly produced vaccines. To this end, the “reverse genetics”technique can be used to engineer mutations or introduce foreignepitopes into the attenuated virus, which would serve as the “parental”strain. In this way, vaccines can be designed for immunization againststrain variants, or in the alternative, against completely differentinfectious agents or disease antigens. For example, the attenuated viruscan be engineered to express neutralizing epitopes of other preselectedstrains. Alternatively, epitopes of viruses other than negative strandRNA viruses can be built into the attenuated mutant virus (e.g., gp160,gp120, or gp41 of HIV). Alternatively, epitopes of non-viral infectiouspathogens (e.g., parasites, bacteria, fungi) can be engineered into thevirus. In yet another alternative, cancer vaccines can be prepared, e.g.by engineering tumor antigens into the attenuated viral backbone.

In a particular embodiment involving RNA viruses with segmented genomes,reassortment techniques can be used to transfer the attenuated phenotypefrom a parental segmented RNA virus strain (a natural mutant, amutagenized virus, or a genetically engineered virus) to a differentvirus strain (a wild-type virus, a natural mutant, a mutagenized virus,or a genetically engineered virus).

The attenuated viruses, which induce robust IFN responses in hosts, mayalso be used in pharmaceutical formulations for the prophylaxis ortreatment of other viral infections, or IFN-treatable diseases, such ascancer. In this regard, the tropism of the attenuated virus can bealtered to target the virus to a desired target organ, tissue or cellsin vivo or ex vivo. Using this approach, the IFN response can be inducedlocally, at the target site, thus avoiding or minimizing the sideeffects of systemic IFN treatments. To this end, the attenuated viruscan be engineered to express a ligand specific for a receptor of thetarget organ, tissue or cells.

The invention is based, in part, on the Applicants' discovery that NS1of wild type influenza virus functions as an IFN antagonist, in that NS1inhibits the IFN mediated response of virus-infected host cells. Viralmutants deficient for NS1 activity were found to be potent inducers ofthe cellular IFN response, and demonstrated.an attenuated phenotype invivo; i.e. the mutant viruses replicate in vivo, but have reducedpathogenic effects. While not intending to be bound to any theory orexplanation for how the invention works, the attenuated features of theviruses of the invention are presumably due to their ability to induce arobust cellular IFN response, and their impaired ability to antagonizethe host IFN response. However, the beneficial features of theattenuated viruses of the invention may not be solely attributable tothe effects on the cellular interferon response. Indeed, alterations inother activities associated with NS1 may contribute to the desiredattenuated phenotype.

The mutant influenza viruses with impaired IFN antagonist activity wereshown to replicate in vivo generating titers that are sufficient toinduce immunological and cytokine responses. For example., vaccinationwith attenuated influenza virus reduced viral titer in animals that weresubsequently challenged with wild-type influenza virus. The attenuatedinfluenza viruses also demonstrated antiviral and antitumor activity.Pre-infection with attenuated influenza virus inhibited replication ofother strains of wild type influenza virus, and other viruses (such asSendai virus) superinfected in embryonated eggs. Inoculation of theattenuated influenza in animals injected with tumor cells reduced thenumber of foci formed. Because influenza virus is known to induce a CTL(cytotoxic T lymphocyte)response, the attenuated virus is a veryattractive candidate for cancer vaccines.

Mutations which diminish but do not abolish the IFN antagonist activityof the virus are preferred for vaccine formulations—such viruses can beselected for growth in both conventional and non-conventionalsubstrates, and for intermediate virulence. In particular, theApplicants have demonstrated that an NS1 C-terminal-truncation mutantreplicates to high titers in IFN deficient substrates, such as 6 and7-day-old embryonated chicken eggs, as well as in the allantoic membraneof 10-day-old embryonated chicken eggs, the conventional substrate forinfluenza virus that does not permit the growth of influenza virusmutants in which the entire NS1 gene is deleted (also referred to hereinas “knockout” mutants). However, replication of the NS1-C terminaltruncation mutant is diminished in 12-day-old embryonated eggs. Thisapproach allows, for the first time, the generation and identificationof live attenuated negative strand RNA viruses that have altered, butnot abolished, IFN antagonist activity, and that are able to grow insubstrates suitable for vaccine preparation. This approach also allowsfor the first time, an efficient selection identification system forinfluenza or other viruses which contain mutations that confer altered,but not abolished, interferon antagonist activity.

The invention also relates to the use of IFN deficient systems topropagate the attenuated viruses that cannot be grown in conventionalsystems currently used for vaccine production. The term “IFN-deficientsystems” as used herein refers to systems, e.g., cells, cell lines andanimals, such as mice, chickens, turkeys, rabbits, rats, etc., which donot produce IFN or produce low levels of IFN, do not respond or respondless efficiently to IFN, and/or are deficient in the activity ofantiviral genes induced by IFN. To this end, Applicants have identifiedor designed a number of IFN-deficient systems that can be used,including but not limited to young embryonated eggs, IFN-deficient celllines (such as VERO cells or genetically engineered cell lines such asSTAT1 knockouts). Alternatively, embryonated eggs or cell lines can bepretreated with compounds that inhibit the IFN system (including drugs,antibodies, antisense, ribozymes, etc.). Yet another embodiment involvesthe use of eggs deficient in the IFN system, e.g., eggs produced bySTAT1 negative birds, especially fowl, including but not limited totransgenic chickens, ducks or turkeys.

4. DESCRIPTION OF THE FIGURES.

FIG. 1. DelNS1 virus inhibits wild-type influenza A virus replication ineggs. Ten-day-old embryonated chicken eggs were inoculated with theindicated pfu of delNS1 virus. Eight hours later, the eggs were infectedwith 10³ pfu of WSN virus. After two days of incubation at 37° C., theallantoic fluid was harvested and WSN virus titers were determined byplaque assay in MDBK cells. Results are the average of two eggs.

FIG. 2. Induction of an antiviral response in embryonated eggs by delNS1virus. Ten-day-old embryonated chicken eggs were inoculated with PBS(untreated) or with 2×10⁴ pfu of delNS1 virus (delNS1 treated). Eighthours later, the eggs were now infected with. 10³ pfu of influenzaA/WSN/33 (H1N1) virus, influenza A/PR8/34 (H+N1) virus, influenza A/X-31(H3N2) virus, influenza B/Lee/40 virus, or Sendai virus. After two daysof incubation, the allantoic fluid was harvested and virus titers weredetermined by a hemagglutination assay. Results are the average of twoeggs.

FIG. 3. CV1 cells were transfected with a plasmid expressing IRF-3 fusedto the green fluorescent protein (GFP). This allowed determining thelocalization of IRF-3 inside the cells by fluorescence microscopy. Insome cases, an NS1 expression plasmid was cotransfected with the IRF-3expression plasmid at the indicated ratios. 24 hours posttransfectioncells were infected at high moi with PR8(WT) or with delNS1 virus asindicated. 10 hours postinfection, cells were analyzed in a fluorescencemicroscope for IRF-3-GFP localization. The percentage of cells showingexclusive cytoplasmic localization (CYT) and both cytoplasmic andnuclear localizations of IRF-3 (Nuc+Cyt) are indicated.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the generation, selection and identification ofattenuated negative strand RNA viruses that have an impaired ability toantagonize the cellular IFN response, and the use of such viruses invaccine and pharmaceutical formulations.

The viruses can have segmented or non-segmented genomes and can beselected from naturally occurring strains, variants or mutants;mutagenized viruses (e.g., by exposure to UV irradiation, mutagens,and/or passaging); reassortants (for viruses with segmented genomes);and/or genetically engineered viruses. For example, the mutant virusescan be generated by natural variation, exposure to UV irradiation,exposure to chemical mutagens, by passaging in non-permissive hosts, byreassortment (i.e., by coinfection of an attenuated segmented virus withanother strain having the desired antigens), and/or by geneticengineering (e.g., using “reverse genetics”). The viruses selected foruse in the invention have defective IFN antagonist activity and areattenuated; i.e., they are infectious and can replicate in vivo, butonly generate low titers resulting in subclinical levels of infectionthat are non-pathogenic. Such attenuated viruses are ideal candidatesfor live vaccines.

In a preferred embodiment, the attenuated viruses selected for use inthe invention should be capable of inducing a robust IFN response in thehost—a feature which contributes to the generation of a strong immuneresponse when used as a vaccine, and which has other biologicalconsequences that make the viruses useful as pharmaceutical agents forthe prevention and/or treatment of other viral infections, or tumorformation in high risk individuals, or other diseases which are treatedwith IFN.

The invention is based, in part, on a number of discoveries andobservations made by the Applicants when working with influenza virusmutants. However, the principles can be analogously applied andextrapolated to other segmented and non-segmented negative strand RNAviruses including, but not limited to paramyxoviruses (Sendai virus,parainfluenza virus, mumps, Newcastle disease virus), morbillivirus(measles virus, canine distemper virus and rinderpest virus);pneumovirus (respiratory syncytial virus and bovine respiratory virus);and rhabdovirus (vesicular stomatitis virus and lyssavirusy.

First, the IFN response is important for containing viral infection invivo. The Applicants found that growth of wild-type influenza virusA/WSN/33 in IFN-deficient mice (STAT1−/− mice) resulted in pan-organinfection; i.e., viral infection was not confined to the lungs as it isin wild-type mice which generate an IFN response (Garcia-Sastre, et al.,1998, J. Virol. 72:8550, which is incorporated by reference herein inits entirety). Second, the Applicants established that NS1 of influenzavirus functions as an IFN antagonist. The Applicants discovered that aninfluenza virus mutant deleted of the entire NS1 gene (i.e., an NS1“knockout”) was not able to grow to high titers in IFN-competent hostcells, and could only be propagated in IFN-deficient hosts. The NS1knockout virus demonstrated an attenuated phenotype (i.e., it was lethalin IFN deficient STAT1−/− mice, but not in wild-type mice) and was foundto be a potent inducer of IFN responses in host cells. (Garcia-Sastre,et al., 1998, Virology 252:324-330, which is incorporated by referenceherein in its entirety). Preinfection with the NS1 knockout mutant virusreduced titers of wild-type influenza and other viruses (e.g., Sendai)superinfected in embryonated eggs. In another experiment, infection withthe NS1 knockout mutant influenza virus reduced foci formation inanimals inoculated with tumor cells. Thus, the NS1 knockout influenzavirus demonstrated interesting biological properties. However, the NS1knockout mutant viruses could not be propagated in conventional systemsfor vaccine production. To overcome this problem, the Applicants usedand developed IFN-deficient systems that allow for production ofreasonable yields of attenuated virus.

In addition, the Applicants designed deletion mutants of NS1, which donot delete the entire gene. Surprisingly, these NS1 mutants were foundto display an “intermediate” phenotype—the virus can be grown inconventional hosts used for propagating influenza virus (although growthis better in the IFN-deficient systems which yield higher titers). Mostimportantly, the deletion mutants are attenuated in vivo, and induce arobust IFN response. Vaccination with the influenza virus NS1 truncatedmutants resulted in low titers of virus in animals subsequentlychallenged with wild-type virus, and afforded protection againstdisease.

The present invention also relates to the substrates designed for theisolation, identification and growth of viruses for vaccine purposes. Inparticular, interferon-deficient substrates for efficiently growinginfluenza virus mutants are described. In accordance with the presentinvention, an interferon-deficient substrate is one that is defective inits ability to produce or respond to interferon. The substrate of thepresent invention may be used for the growth of any number of viruseswhich may require interferon-deficient growth environment. Such virusesmay include, but are not limited to paramyxoviruses (Sendai virus,parainfluenza virus, mumps, Newcastle disease virus), morbillivirus(measles virus, canine distemper virus and rinderpest virus);pneumovirus (respiratory syncytial virus and bovine respiratory virus);rhabdovirus (vesicular stomatitis virus and lyssavirus).

The invention also relates to the use of the attenuated virus of theinvention in vaccines and pharmaceutical preparations for humans oranimals. In particular, the attenuated viruses can be used as vaccinesagainst a broad range of viruses and/or antigens, including but notlimited to antigens of strain variants, different viruses or otherinfectious pathogens (e.g., bacteria, parasites, fungi), or tumorspecific antigens. In another embodiment, the attenuated viruses, whichinhibit viral replication and tumor formation, can be used for theprophylaxis or treatment of infection (viral or nonviral pathogens) ortumor formation or treatment of diseases for which IFN is of therapeuticbenefit. Many methods may be used to introduce the live attenuated virusformulations to a human or animal subject to induce an immune orappropriate cytokine response. These include, but are not limited to,intranasal, intratrachial, oral, intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous routes. In a preferredembodiment, the attenuated viruses of the present invention areformulated for delivery intranasally.

5.1 Generation of Mutants with Altered IFN Antagonist Activity

Any mutant virus or strain which has a decreased IFN antagonist activitycan be selected and used in accordance with the invention. In oneembodiment, naturally occurring mutants or variants, or spontaneousmutants can be selected that have an impaired ability to antagonize thecellular IFN response. In another embodiment, mutant viruses can begenerated by exposing the virus to mutagens, such as ultravioletirradiation or chemical mutagens, or by multiple passages and/or passagein non-permissive hosts. Screening in a differential growth system canbe used to select for those mutants having impaired IFN antagonistfunction. For viruses with segmented genomes, the attenuated phenotypecan be transferred to another strain having a desired antigen byreassortment, (i.e., by coinfection of the attenuated virus and thedesired strain, and selection for reassortants displaying bothphenotypes).

In another embodiment, mutations can be engineered into a negativestrand RNA virus such as influenza, RSV, NDV, VSV and PIV, using“reverse genetics” approaches. In this way, natural or other mutationswhich confer the attenuated phenotype can be engineered into vaccinestrains. For example, deletions, insertions or substitutions of thecoding region of the gene responsible for IFN antagonist activity (suchas the NS1 of influenza) can be engineered. Deletions, substitutions orinsertions in the non-coding region of the gene responsible for IFNantagonist activity are also contemplated. To this end, mutations in thesignals responsible for the transcription, replication, polyadenylationand/or packaging of the gene responsible or the IFN-antagonist activitycan be engineered. For example, in influenza, such modifications caninclude but are not limited to: substitution of the non-coding regionsof an influenza A virus gene by the non-coding regions of an influenza Bvirus gene (Muster, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:5177),base pairs exchanges in the non-coding regions of an influenza virusgene (Fodor, et al., 1998, J Virol. 72:6283), mutations in the promoterregion of an influenza virus gene (Piccone, et al., 1993, Virus Res.28:99; Li, et al., 1992, J Virol. 66:4331), substitutions and deletionsin the stretch of uridine residues at the 5′ end of an influenza virusgene affecting polyadenylation (Luo, et al., 1991, J Virol. 65:2861; Li,et al., J Virol. 1994, 68:1245). Such mutations, for example to thepromoter, could down-regulate the expression of the gene responsible forIFN antagonist activity. Mutations in viral genes which may regulate theexpression of the gene responsible for IFN antagonist activity are alsowithin the scope of viruses that can be used in accordance with theinvention.

The present invention also relates to mutations to the NS1 gene segmentthat may not result in an altered IFN antagonist activity or anIFN-inducing phenotype but rather results in altered viral functions andan attenuated phenotype e.g., altered inhibition of nuclear export ofpoly(A)-containing mRNA, altered inhibition of pre-mRNA splicing,altered inhibition of the activation of PKR by sequestering of dsRNA,altered effect on translation of viral RNA and altered inhibition ofpolyadenylation of host mRNA (e.g., see Krug in Textbook of Influenza,Nicholson et al. Ed. 1998, 82-92, and references cited therein).

The reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative strand virus RNA which are essential for the recognition byviral polymerases and for packaging signals necessary to generate amature virion. The recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. A more efficient transfection is achieved if theviral polymerase proteins are present during transcription of thesynthetic RNAs either in vitro or in vivo. The synthetic recombinantRNPs can be rescued into infectious virus particles. The foregoingtechniques are described in U.S. Pat. No. 5,166,057 issued Nov. 24,1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in EuropeanPatent Publication EP 0702085A1, published Feb. 20, 1996; in U.S. patentapplication Ser. No. 09/152,845; in International Patent PublicationsPCT WO97/12032 published Apr. 3, 1997; WO96/34625 published Nov. 7,1996; in European Patent Publication EP-A780475; WO 99/02657 publishedJan. 21, 1999; WO 98/53078 published Nov. 26, 1998; WO 98/02530published Jan. 22, 1998; WO 99/15672 published Apr. 1, 1999; WO 98/13501published Apr. 2, 1998; WO 97/06270 published Feb. 20, 1997; and EPO 780475A1 published Jun. 25, 1997, each of which is incorporated byreference herein in its entirety.

Attenuated viruses generated by the reverse genetics approach can beused in the vaccine and pharmaceutical formulations described herein.Reverse genetics techniques can also be used to engineer additionalmutations to other viral genes important for vaccine production—i.e.,the epitopes of useful vaccine strain variants can be engineered intothe attenuated virus. Alternatively, completely foreign epitopes,including antigens derived from other viral or non-viral pathogens canbe engineered into the attenuated strain. For example, antigens ofnon-related viruses such as HIV (gp160, gp120, gp41) parasite antigens(e.g., malaria), bacterial or fungal antigens or tumor antigens can beengineered into the attenuated strain. Alternatively, epitopes whichalter the tropism of the virus in vivo can be engineered into thechimeric attenuated viruses of the invention.

In an alternate embodiment, a combination of reverse genetics techniquesand reassortant techniques can be used to engineer attenuated viruseshaving the desired epitopes in segmented RNA viruses. For example, anattenuated virus (generated by natural selection, mutagenesis or byreverse genetics techniques) and a strain carrying the desired vaccineepitope (generated by natural selection, mutagenesis or by reversegenetics techniques) can be co-infected in hosts that permitreassortment of the segmented genomes. Reassortants that display boththe attenuated phenotype and the desired epitope can then be selected.

In another embodiment, the virus to be mutated is a DNA virus (e.g.,vaccinia, adenovirus, baculovirus) or a positive strand RNA virus (e.g.,polio virus). In such cases, recombinant DNA techniques which are wellknown in the art may be used (e.g., see U.S. Pat. No. 4,769,330 toPaoletti, U.S. Pat. No. 4,215,051 to Smith each of which is incorporatedherein by reference in its entirety).

Any virus may be engineered in accordance with the present invention,including but not limited to the families set forth in Table 2 below.

TABLE 2 FAMILIES OF HUMAN AND ANIMAL VIRUSES VIRUS CRARACTERISTICS VIRUSFAMILY dsDNA Enveloped Poxviridae Irididoviridae HerpesviridaeNonenveloped Adenoviridae Papovaviridae Hepadnaviridae ssDNANonenveloped Parvoviridae dsRNA Nonenveloped Reoviridae BirnaviridaessRNA Enveloped Positive-Sense Genome No DNA Step in ReplicationTogaviridae Flaviviridae Coronaviridae Hepatitis C Virus DNA Step inReplication Retroviridae Negative-Sense Genome Non-Segmented GenomeParamyxoviridae Rhabdoviridae Filoviridae Segmented GenomeOrthomyxoviridae Bunyaviridae Arenaviridae Nonenveloped PicornaviridaeCaliciviridae Abbreviations used: ds = double stranded; ss = singlestranded; enveloped = possessing an outer lipid bilayer derived from thehost cell membrane; positive-sense genome = for RNA viruses, genomesthat are composed of nucleotide sequences that are directly translatedon ribosomes, = for DNA viruses, genomes that are composed of nucleotidesequences that are the same as the mRNA; negative-sense genome = genomes# that are composed of nucleotide sequences complementary to thepositive-sense strand.

In a preferred embodiment, the present invention relates to geneticallyengineered influenza viruses containing deletions and/or truncations ofthe NS1 gene product. NS1 mutants of influenza A and B are particularlypreferred. In one approach, portions of the amino terminal region of theNS1 gene product are retained whereas portions of the C-terminal regionof the NS1 gene product are deleted. Specific desired mutations can beengineered by way of nucleic acid insertion, deletion, or mutation atthe appropriate codon. In particular, the truncated NS1 proteins havefrom 1-60 amino acids, 1-70 amino acids, 1-80 amino acids, 1-90 aminoacids (the N-terminal amino acid is 1), and preferably 90 amino acids;from 1-100 amino acids, and preferably 99 amino acids; from 1-110 aminoacids; from 1-120 amino acids; or from 1-130 amino acids, and preferably124 amino acids of the wildtype NS1 gene product.

The present invention also relates to any genetically engineeredinfluenza virus in which the NS1 gene product has been modified bytruncation or modification of the NS1 protein that confers upon themutant viruses the following phenotypes: the ability of the viruses togrow to high titers in unconventional substrates, such as 6-7 day oldchicken eggs, or the ability of the viruses to induce a host interferonresponse. For influenza A viruses, these include, but are not limitedto: viruses having an NS1 truncations.

The present invention includes the use of naturally occurring mutantinfluenza viruses A or B having the attenuated phenotype, as well asinfluenza virus strains engineered to contain such mutations responsiblefor the attenuated phenotype. For influenza A viruses, these include,but are not limited to: viruses having an NS1 of 124 amino acids (Nortonet al., 1987, Virology 156:204-213, which is incorporated by referenceherein in its entirety). For influenza B viruses, these include, but arenot limited to: viruses having an NS1 truncation mutant comprising 127amino acids derived from the N-terminus (B/201) (Norton et al., 1987,Virology 156:204-213, which is incorporated by reference herein in itsentirety), and viruses having an NS1 truncation mutant comprising 90amino acids derived from the N-terminus (B/AWBY-234) (Tobita et al.,1990, Virology 174:314-19, which is incorporated by reference herein inits entirety). The present invention encompasses the use of naturallyoccurring mutants analogous to NS1/38, NS1/80, NS1/124, (Egorov, et al.,1998, J. Virol. 72(8):6437-41) as well as the naturally occurringmutants, A/Turkey/ORE/71, B/201 or B/AWBY-234.

The attenuated influenza virus may be further engineered to expressantigens of other vaccine strains (e.q., using reverse genetics orreassortment). Alternatively, the attenuated influenza viruses may beengineered, using reverse genetics or reassortment with geneticallyengineered viruses, to express completely foreign epitopes, e.g.,antigens of other infectious pathogens, tumor antigens, or targetingantigens. Since the NS RNA segment is the shortest among the eight viralRNAs, it is possible that the NS RNA will tolerate longer insertions ofheterologous sequences than other viral genes. Moreover, the NS RNAsegment directs the synthesis of high levels of protein in infectedcells, suggesting that it would be an ideal segment for insertions offoreign antigens. However, in accordance with the present invention, anyone of the eight segments of influenza viruses may be used for theinsertion of heterologous sequences. For example, where surface antigenpresentation is desired, segments encoding structural proteins, e.g., HAor NA may be used.

5.2 Host-Restricted Based Selection System

The invention encompasses methods of selecting viruses which have thedesired phenotype, i.e., viruses which have low or no IFN antagonistactivity, whether obtained from natural variants, spontaneous variants(i.e., variants which evolve during virus propagation), mutagenizednatural variants, reassortants and/or genetically engineered viruses.Such viruses can be best screened in differential growth assays thatcompare growth in IFN-deficient versus IFN-competent host systems.Viruses which demonstrate better growth in the IFN-deficient systemsversus IFN competent systems are selected; preferably, viruses whichgrow to titers at least one log greater in IFN-deficient systems ascompared to an IFN-competent system are selected.

Alternatively, the viruses can be screened using IFN assay systems e.g.,transcription based assay systems in which reporter gene expression iscontrolled by an IFN-responsive promoter. Reporter gene expression ininfected versus uninfected cells can be measured to identify viruseswhich efficiently induce an IFN response, but which are unable toantagonize the IFN response. In a preferred embodiment, however,differential growth assays are used to select viruses having the desiredphenotype, since the host system used (IFN-competent versusIFN-deficient) applies the appropriate selection pressure.

For example, growth of virus (as measured by titer) can be compared in avariety of cells, cell lines, or animal model systems that express IFNand the components of the IFN response, versus cells, cell lines, oranimal model systems deficient for IFN or components of the IFNresponse. To this end, growth of virus in cell lines as VERO cells(which are IFN deficient) versus MDCK cells (which are IFN-competent)can be compared. Alternatively, IFN-deficient cell lines can be derivedand established from animals bred or genetically engineered to bedeficient in the IFN system (e.g., STAT1−/− mutant mice). Growth ofvirus in such cell lines, as compared to IFN-competent cells derived,for example, from wild-type animals (e.g., wild-type mice) can bemeasured. In yet another embodiment, cell lines which are IFN-competentand known to support the growth of wild type virus can be engineered tobe IFN-deficient, (e.g., by knocking out STAT1, IRF3, PKR, etc.)Techniques which are well known in the art for the propagation ofviruses in cell lines can be used (see, for example, the workingexamples infra). Growth of virus in the standard IFN competent cell lineversus the IFN deficient genetically engineered cell line can becompared.

Animal systems can also be used. For example, for influenza, growth inyoung, IFN-deficient embryonated eggs, e.g., about 6 to about 8 daysold, can be compared to growth in older, IFN-competent eggs, e.g. about10 to 12 days old. To this end, techniques well known in the art forinfection and propagation in eggs can be used (e.g., see workingexamples, infra). Alternatively, growth in IFN-deficient STAT1−/− micecan be compared to IFN-competent wild type mice. In yet anotheralternative, growth in IFN-deficient embryonated eggs produced by, forexample, STAT1−/− transgenic fowl can be compared to growth inIFN-competent eggs produced by wild-type fowl.

For purposes of screening, however, transient IFN-deficient systems canbe used in lieu of genetically manipulated systems. For example, thehost system can be treated with compounds that inhibit IFN productionand/or components of the IFN response (e.g., drugs, antibodies againstIFN, antibodies against IFN-receptor, inhibitors of PKR, antisensemolecules and ribozymes, etc.). Growth of virus can be compared inIFN-competent untreated controls versus IFN-deficient treated systems.For example, older eggs which are IFN-competent can be pretreated withsuch drugs prior to infection with the virus to be screened. Growth iscompared to that achieved in untreated control eggs of the same age.

The screening methods of the invention provide a simple and easy screento identify mutant viruses with abolished IFN antagonist activity by theinability of the mutant virus to grow in IFN-responsive environments, ascompared to the ability of the mutant virus to grow in IFN-deficientenvironments. The screening methods of the invention may also be used toidentify mutant viruses with altered, but not abolished IFN antagonistactivity by measuring the ability of the mutant virus to grow in bothIFN-responsive e.g., 10-day old embryonated eggs or MDCK cells andIFN-deficient environments e.a., 6-to-7-day old embryonated eggs or Verocells. For example, influenza viruses showing at least one log lowertiters in 10-days-old eggs versus 6-7 days old eggs will be consideredimpaired in their ability to inhibit the IFN response. In anotherexample, influenza viruses showing at least one log lower titer in 12day old eggs (which mount a high IFN response) versus 10 day old eggs(which mount a moderate IFN response) are considered partially impairedin their ability to antagonize the IFN response, and are consideredattractive vaccine candidates.

The selection methods of the invention also encompass identifying thosemutant viruses which induce IFN responses. In accordance with theselection methods of the invention, induction of IFN responses may bemeasured by assaying levels of IFN expression or expression of targetgenes or reporter genes induced by IFN following infection with themutant virus or activation of transactivators involved in the IFNexpression and/or the IFN response.

In yet another embodiment of the selection systems of the invention,induction of IFN responses may be determined by measuring thephosphorylated state of components of the IFN pathway followinginfection with the test mutant virus, e.g., IRF-3, which isphosphorylated in response to double-stranded RNA. In response to type IIFN, Jak1 kinase and TyK2 kinase, subunits of the IFN receptor, STAT1,and STAT2 are rapidly tyrosine phosphorylated. Thus, in order todetermine whether the mutant virus induces IFN responses, cells, such as293 cells, are infected with the test mutant virus and followinginfection, the cells are lysed. IFN pathway components, such as Jak1kinase or TyK2 kinase, are immunoprecipitated from the infected celllysates, using specific polyclonal sera or antibodies, and the tyrosinephosphorylated state of the kinase is determined by immunoblot assayswith an anti-phosphotyrosine antibody (e.g., see Krishnan et al. 1997,Eur. J. Biochem. 247: 298-305). An enhanced phosphorylated state of anyof the components of the IFN pathway following infection with the mutantvirus would indicate induction of IFN responses by the mutant virus.

In yet another embodiment, the selection systems of the inventionencompass measuring the ability to bind specific DNA sequences or thetranslocation of transcription factors induced in response to viralinfection, e.g., IRF3, STAT1, STAT2, etc. In particular, STAT1 and STAT2are phosphorylated and translocated from the cytoplasm to the nucleus inresponse to type I IFN. The ability to bind specific DNA sequences orthe translocation of transcription factors can be measured by techniquesknown to those of skill in the art, e.g., electromobility gel shiftassays, cell staining, etc.

In yet another embodiment of the selection systems of the invention,induction of IFN responses may be determined by measuring IFN-dependenttranscriptional activation following infection with the test mutantvirus. In this embodiment, the expression of genes known to be inducedby IFN, e.g., Mx, PKR, 2-5-oligoadenylatesynthetase, majorhistocompatibility complex (MHC) class I, etc., can be analyzed bytechniques known to those of skill in the art (e.g., northern blots,western blots, PCR, etc.). Alternatively, test cells such as humanembryonic kidney cells or human osteogenic sarcoma cells, are engineeredto transiently or constitutively express reporter genes such asluciferase reporter gene or chloramphenicol transferase (CAT) reportergene under the control of an interferon stimulated response element,such as the IFN-stimulated promoter of the ISG-54K gene (Bluyssen etal., 1994, Eur. J. Biochem. 220:395-402). Cells are infected with thetest mutant virus and the level of expression of the reporter genecompared to that in uninfected cells or cells infected with wild-typevirus. An increase in the level of expression of the reporter genefollowing infection with the test virus would indicate that the testmutant virus is inducing an IFN response.

In yet another embodiment, the selection systems of the inventionencompass measuring IFN induction by determining whether an extract fromthe cell or egg infected with the test mutant virus is capable ofconferring protective activity against viral infection. Morespecifically, groups of 10-day old embryonated chicken eggs are infectedwith the test mutant virus or the wild-type virus. Approximately 15 to20 hours post infection, the allantoic fluid is harvested and tested forIFN activity by determining the highest dilution with protectiveactivity against VSV infection in tissue culture cells, such as CEFcells.

5.3 Propagation of Virus in Interferon Deficient Growth Substrates

The present invention relates to novel methods and substrates for thepropagation of viruses. The invention relates to IFN-deficientsubstrates and methods for propagating viruses in these substrates. Inparticular, the invention relates to methods of propagating viruses inimmature embryonated eggs, e.g., up to, but less than ten-day-old eggs,preferably six to nine day old eggs, which are normally not used forgrowing viruses due to their fragile condition and smaller allantoiccavity. In accordance with the present invention, immature embryonatedeggs encompass eggs which as a course of nature are up to, but less thanten-day-old eggs, preferably six- to nine-day-old eggs. Immatureembryonated eggs of the present invention also encompass eggs whichartificially mimic immature eggs up to, but less than ten-day-old, as aresult of alterations to the growth conditions, e.g., changes inincubation temperatures; treating with drugs; or any other alterationwhich results in an egg with a retarded development, such that the IFNsystem is not fully developed as compared with ten- to twelve-day-oldeggs. The invention relates to methods of growing any virus in theseunconventional growth substrates, including, but not limited to,negative strand RNA viruses, including segmented and non segmentedviruses, such as paramyxoviruses (e.g., Newcastle disease virus, PIV)rhabdoviruses, rabies, orthomyxoviruses (e.g., influenza A and B),bunyaviruses, arenaviruses; positive strand RNA viruses, (e.g.,retroviruses); DNA viruses, (e.g., poxviruses)

In accordance with the methods of the present invention, the viruseswhich may be grown in immature embryonated eggs are selected fromnaturally occurring strains, variants or mutants, mutagenized virus,reassortants and/or genetically engineered viruses. The methods of thepresent invention encompass growing the viruses in up to ten-day-oldeggs, six to nine-day old eggs, preferably using appropriate growthconditions, (see e.g., growth conditions set forth in Section 6 below)and collecting the progeny virus. The methods of the inventionencompassing growth of viruses in immature embryonated eggs, preferablysix to nine-day-old eggs, are particularly attractive for growingviruses suitable for use in vaccine formulations.

The invention also encompasses methods and IFN-deficient substrates forthe growth and isolation of naturally occurring or engineered mutantviruses having altered IFN antagonist activity. IFN-deficient substrateswhich can be used to support the growth of the attenuated mutant virusesinclude, but are not limited to naturally occurring cells, cell lines,animals or embryonated eggs that are IFN deficient, e.g., Vero cells,young embryonated eggs; recombinant cells or cell lines that areengineered to be IFN deficient, e.q., IFN-deficient cell lines derivedfrom STAT1 knockout mice or other similarly engineered transgenicanimals; embryonated eggs obtained from IFN deficient birds, especiallyfowl (e.g., chickens, ducks, turkeys) including flock that are bred tobe IFN-deficient or transgenic birds (e.g., STAT1 knockouts).Alternatively, the host system, cells, cell lines, eggs or animals canbe genetically engineered to express transgenes encoding inhibitors ofthe IFN system, e.g., dominant-negative mutants, such as STAT1 lackingthe DNA binding domain, antisense RNA, ribozymes, inhibitors of IFNproduction, inhibitors of IFN signaling, and/or inhibitors of antiviralgenes induced by IFN. It should be recognized that animals that are bredor genetically engineered to be IFN deficient will be somewhatimmunocompromised, and should be maintained in a controlled, diseasefree environment. Thus, appropriate measures (including the use ofdietary antibiotics) should be taken to limit the risk of exposure toinfectious agents of transgenic IFN deficient animals, such as flocks ofbreeding hens, ducks, turkeys etc. Alternatively, the host system, e.g.,cells, cell lines, eggs or animals can be treated with a compound whichinhibits IFN production and/or the IFN pathway e.g., drugs, antibodies,antisense molecules, ribozyme molecules targeting the STAT1 gene, and/orantiviral genes induced by IFN.

In accordance with the present invention, immature embryonated chickeneggs encompass eggs which as a course of nature are up to but not yetten-day-old eggs, preferably six-to nine-day-old eggs; and eggs whichartificially mimic immature eggs up to, but not yet ten-day-old, as aresult of alterations to the growth conditions, e.g., changes inincubation temperatures; treating with drugs; or any other alterationwhich results in an egg with a retarded development, such that the IFNsystem of the egg is not fully developed as compared to 10- to12-day-old eggs.

5.3.1 Natural IFN Deficient Substrates

In one embodiment, the present invention relates to growing naturallyoccurring and engineered mutant viruses in unconventional substrates,such as immature embryonated eggs which have not yet developed an IFNsystem. Immature embryonated eggs are normally not used to grow virusdue to their fragile condition and smaller allantoic volume. The presentinvention encompasses growing mutant viruses in embryonated eggs lessthan 10 days old; preferably growing mutated virus in 8-day oldembryonated eggs and most preferably, in 6 to 8-day old eggs.

The present invention also encompasses methods of growing and isolatingmutated viruses having altered IFN antagonist activity in cells and celllines which naturally do not have an IFN pathway or have a deficient IFNpathway or have a deficiency in the IFN system e.g., low levels of IFNexpression as compared to wild-type cells. In a particular preferredembodiment, the present invention relates to methods of growing mutatedviruses having an altered IFN antagonist activity in Vero cells.

5.3.2 Genetically Engineered IFN Deficient Substrates

The present invention relates to methods of growing and isolatingmutated viruses having altered IFN antagonist activity in a geneticallyengineered IFN deficient substrate. The present invention encompassestransgenic avians in which a gene essential to the IFN system ismutated, e.g., STAT1, which would lay eggs that are IFN deficient. Thepresent invention further encompasses avian transgenics which expressdominant-negative transcription factors, e.g., STAT1 lacking the DNAbinding domain, ribozymes, antisense RNA, inhibitors of IFN production,inhibitors of IFN signaling, and inhibitors of antiviral genes inducedin response to IFN. The benefit of using eggs from an IFN-deficienttransgenic avian is that the conventional 10 day age eggs may be used togrow the virus which are more stable and have a larger volume due totheir larger size. In yet another embodiment, cell lines may begenetically engineered to be IFN deficient. The present inventionencompasses cell lines in which a gene essential to the IFN synthesis,IFN pathway, and/or an antiviral gene(s) induced by IFN are mutated,e.g., STAT1.

The invention provides recombinant cell lines or animals, in particularavians, in which one or more genes essential for the IFN pathway, e.g.interferon receptor, STAT1 etc. has been disrupted, i.e., is a“knockout”; the recombinant animal can be any animal but in a preferredembodiment is an avian, e.g. chicken, turkey, hen, duck, etc. (see,e.g., Sang, 1994, Trends Biotechnol. 12:415; Perry, et al., 1993,Transgenic Res. 2:125; Stern, C. D., 1996, Curr Top Microbiol Immunol212:195-206; and Shuman, 1991, Experientia 47:897 for reviews regardingthe production of avian transgenics each of which is incorporated byreference herein in its entirety). Such a cell line or animal can begenerated by any method known in the art for disrupting a gene on thechromosome of the cell or animal. Such techniques include, but are notlimited to pronuclear microinjection (Hoppe & Wagner, 1989, U.S. Pat.No. 4,873,191); retrovirus mediated gene transfer into germ lines (Vander Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717); etc.For a review of such techniques, see Gordon, 1989, Transgenic Animals,Intl. Rev. Cytol. 115:171, which is incorporated by reference herein inits entirety.

In particular, a STAT1 knockout animal can be produced by promotinghomologous recombination between a STAT1 gene in its chromosome and anexogenous STAT1 gene that has been rendered biologically inactive(preferably by insertion of a heterologous sequence, e.g., an antibioticresistance gene). Homologous recombination methods for disrupting genesin the mouse genome are described, for example, in Capecchi (1989,Science 244:1288) and Mansour et al. (1988, Nature 336:348-352).

Briefly, all or a portion of a STAT1 genomic clone is isolated fromgenomic DNA from the same species as the knock-out cell or animal. TheSTAT1 genomic clone can be isolated by any method known in the art forisolation of genomic clones (e.g. by probing a genomic library with aprobe derived from a STAT1 sequence such as those sequences provided insee Meraz et al. 1996, Cell 84: 431-442; Durbin et al. 1996, Cell:84443-450, and references cited therein). Once the genomic clone isisolated, all or a portion of the clone is introduced into a recombinantvector. Preferably, the portion of the clone introduced into the vectorcontains at least a portion of an exon of the STAT1 gene, i.e., containsa STAT1 protein coding sequence. A sequence not homologous to the STAT1sequence, preferably a positive selectable marker, such as a geneencoding an antibiotic resistance gene, is then introduced into theSTAT1 gene exon. The selectable marker is preferably operably linked toa promoter, more preferably a constitutive promoter. The non-homologoussequence is introduced anywhere in the STAT1 coding sequence that willdisrupt STAT1 activity, e.g., at a position where point mutations orother mutations have been demonstrated to inactivate STAT1 proteinfunction. For example, but not by way of limitation, the non-homologoussequence can be inserted into the coding sequence for the portion of theSTAT1 protein containing all or a portion of the kinase domain (e.g.,the nucleotide sequence coding for at least 50, 100, 150, 200 or 250amino acids of the kinase domain).

The positive selectable marker is preferably a neomycin resistance gene(neo gene) or a hygromycin resistance gene (hygro gene). The promotermay be any promoter known in the art; by way of example the promoter maybe the phosphoglycerate kinase (PGK) promoter (Adra et al.,1987, Gene60:65-74), the PolII promoter (Soriano et al., 1991. Cell 64:693-701),or the MC1 promoter, which is a synthetic promoter designed forexpression in embryo-derived stem cells (Thomas & Capecchi, 1987, Cell51:503-512). Use of a selectable marker, such as an antibioticresistance gene, allows for the selection of cells that haveincorporated the targeting vector (for example, the expression of theneo gene product confers resistance to G418, and expression of the hygrogene product confers resistance to hygromycin).

In a preferred embodiment, a negative selectable marker for acounterselection step for homologous, as opposed to non-homologous,recombination of the vector is inserted outside of the STAT1 genomicclone insert. For example, such a negative selectable marker is the HSVthymidine kinase gene (HSV-tk), the expression of which makes cellssensitive to ganciclovir. The negative selectable marker is preferablyunder the control of a promoter such as, but not limited to the PGKpromoter, the PolII promoter or the MC1 promoter.

When homologous recombination occurs, the portions of the vector thatare homologous to the STAT1 gene, as well as the non-homologous insertwithin the STAT1 gene sequences, are incorporated into the STAT1 gene inthe chromosome, and the remainder of the vector is lost. Thus, since thenegative selectable marker is outside the region of homology with theSTAT1 gene, cells in which homologous recombination has occurred (ortheir progeny), will not contain the negative selectable marker. Forexample, if the negative selectable marker is the HSV-tk gene, the cellsin which homologous recombination has occurred will not expressthymidine kinase and will survive exposure to ganciclovir. Thisprocedure permits the selection of cells in which homologousrecombination has occurred, as compared to non-homologous recombinationin which it is likely that the negative selectable marker is alsoincorporated into the genome along with the STAT1 sequences and thepositive selectable marker. Thus, cells in which non-homologousrecombination has occurred would most likely express thymidine kinaseand be sensitive to ganciclovir.

Once the targeting vector is prepared, it is linearized with arestriction enzyme for which there is a unique site in the targetingvector, and the linearized vector is introduced into embryo-derived stem(ES) cells (Gossler et al., 1986, Proc. Natl. Acad. Sci. USA83:9065-9069) by any method known in the art, for example byelectroporation. If the targeting vector includes a positive selectablemarker and a negative, counterselectable marker, the ES cells in whichhomologous recombination has occurred can be selected by incubation inselective media. For example, if the selectable markers are the neoresistance gene and the HSV-tk gene, the cells are exposed to G418(e.g., approximately 300 μg/ml) and ganciclovir (e.g., approximately 2μM).

Any technique known in the art for genotyping, for example but notlimited to Southern blot analysis or the polymerase chain reaction, canbe used to confirm that the disrupted STAT1 sequences have homologouslyrecombined into the STAT1 gene in the genome of the ES cells. Becausethe restriction map of the STAT1 genomic clone is known and the sequenceof the STAT1 coding sequence is known(see Meraz et al. 1996, Cell84:431, Durbin et al. 1996, Cell 84:443-450, all references citedtherein.), the size of a particular restriction fragment or a PCRamplification product generated from DNA from both the disrupted andnon-disrupted alleles can be determined. Thus, by assaying for arestriction fragment or PCR product, the size of which differs betweenthe disrupted and non-disrupted STAT1 gene, one can determine whetherhomologous recombination has occurred to disrupt the STAT1 gene.

The ES cells with the disrupted STAT1 locus can then be introduced intoblastocysts by microinjection and then the blastocysts can be implantedinto the uteri of pseudopregnant mice using routine techniques. Theanimal that develop from the implanted blastocysts are chimeric for thedisrupted allele. The chimeric males can be crossed to females, and thiscross can be designed such that germline transmission of the allele islinked to transmission of a certain coat color. The germlinetransmission of the allele can be confirmed by Southern blotting or PCRanalysis, as described above, of genomic DNA isolated from tissuesamples.

5.3.3 Transient IFN Deficient Substrates

The cells, cell lines, animals or eggs can be pre-treated with compoundsthat inhibit the IFN system. In accordance with the present invention,compounds which inhibit synthesis of IFN, or the activity or theexpression of the components of the IFN system can be used to pretreathosts, e.g., compounds that inhibit the synthesis of IFN, the activityof IFN, the IFN receptor, other targets in the IFN signal transductionpathway, or that inhibit the activity of antiviral genes induced by IFN.Examples of compounds which may be used in accordance with the presentinvention, include, but are not limited to, nucleic acid molecules,antibodies, peptides, antagonists of the IFN receptor, inhibitors of theSTAT1 pathway, inhibitors of PKR, etc. In accordance with the presentinvention, nucleic acid molecules include antisense molecules, ribozymesand triple helix molecules that target genes encoding essentialcomponents of the IFN system, e.g., STAT1. Nucleic acid molecules alsoencompass nucleotides encoding dominant negative mutants of componentsof the IFN system; e.g. prior to infection with the viral mutant, thecells can be transfected with a DNA encoding a truncated, signallingincompetent mutant of the IFN receptor.

Dominant-negative mutants which may be used in accordance with thepresent invention to inhibit the IFN pathway include kinase deficientversions of Jak1, TyK2 or transcription factors lacking DNA bindingdomains STAT1, and STAT2 (see, e.g., Krishnan et al., 1997, Eur. J.Biochem. 247: 298-305)

5.4 Vaccine Formulations

The invention encompasses vaccine formulations comprising the attenuatednegative strand RNA viruses having an impaired ability to antagonize thecellular IFN response, and a suitable excipient. The virus used in thevaccine formulation may be selected from naturally occurring mutants orvariants, mutagenized viruses or genetically engineered viruses.Attenuated strains of segmented RNA viruses can also be generated viareassortment techniques, or by using a combination of the reversegenetics approach and reassortment techniques. Naturally occurringvariants include viruses isolated from nature as well as spontaneousoccurring variants generated during virus propagation, having animpaired ability to antagonize the cellular IFN response. The attenuatedvirus can itself be used as the active ingredient in the vaccineformulation. Alternatively, the attenuated virus can be used as thevector or “backbone” of recombinantly produced vaccines. To this end,recombinant techniques such as reverse genetics (or, for segmentedviruses, combinations of the reverse genetics and reassortmenttechniques) may be used to engineer mutations or introduce foreignantigens into the attenuated virus used in the vaccine formulation. Inthis way, vaccines can be designed for immunization against strainvariants, or in the alternative, against completely different infectiousagents or disease antigens.

Virtually any heterologous gene sequence may be constructed into theviruses of the invention for use in vaccines. Preferably, epitopes thatinduce a protective immune response to any of a variety of pathogens, orantigens that bind neutralizing antibodies may be expressed by or aspart of the viruses. For example, heterologous gene sequences that canbe constructed into the viruses of the invention for use in vaccinesinclude but are not limited to epitopes of human immunodeficiency virus(HIV) such as gp120; hepatitis B virus surface antigen (HBsAg); theglycoproteins of herpes virus (eq. gD, gE); VP1 of poliovirus; antigenicdeterminants of non-viral pathogens such as bacteria and parasites, toname but a few. In another embodiment, all or portions of immunoglobulingenes may be expressed. For example, variable regions of anti-idiotypicimmunoglobulins that mimic such epitopes may be constructed into theviruses of the invention. In yet another embodiment, tumor associatedantigens may be expressed.

Either a live recombinant viral vaccine or an inactivated recombinantviral vaccine can be formulated. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

Vaccine formulations may include genetically engineered negative strandRNA viruses that have mutations in the NS1 or analogous gene includingbut not limited to the truncated NS1 influenza mutants described in theworking examples, infra. They may also be formulated using naturalvariants, such as the A/turkey/Ore/71 natural variant of influenza A, orB/201, and B/AWBY-234, which are natural variants of influenza B. Whenformulated as a live virus vaccine, a range of about 10⁴ pfu to about5×10⁶ pfu per dose should be used.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to intranasal, intratracheal,oral, intradermal, intramuscular, intraperitoneal, intravenous, andsubcutaneous routes. It may be preferable to introduce the virus vaccineformulation via the natural route of infection of the pathogen for whichthe vaccine is designed, or via the natural route of infection of theparental attenuated virus. Where a live influenza virus vaccinepreparation is used, it may be preferable to introduce the formulationvia the natural route of infection for influenza virus. The ability ofinfluenza virus to induce a vigorous secretory and cellular immuneresponse can be used advantageously. For example, infection of therespiratory tract by influenza viruses may induce a strong secretoryimmune response, for example in the urogenital system, with concomitantprotection against a particular disease causing agent.

A vaccine of the present invention, comprising 10⁴-5×10⁶ pfu of mutantviruses with altered IFN antagonist activity, could be administeredonce. Alternatively, a vaccine of the present invention, comprising10⁴-5×10⁶ pfu of mutant viruses with altered IFN antagonist activity,could be administered twice or three times with an interval of 2 to 6months between doses. Alternatively, a vaccine of the present invention,comprising 10⁴-5×10⁶ pfu of mutant viruses with altered IFN antagonistactivity, could be administered as often as needed to an animal,preferably a mammal, and more preferably a human being.

5.5 Pharmaceutical Compositions

The present invention encompasses pharmaceutical compositions comprisingmutant viruses with altered IFN antagonist activity to be used asanti-viral agents or anti-tumor agents or as agents againstIFN-sensitive diseases. The pharmaceutical compositions have utility asan anti-viral prophylactic and may be administered to an individual atrisk of getting infected or is expected to be exposed to a virus. Forexample, in the event that a child comes home from school where he isexposed to several classmates with the flu, a parent would administerthe anti-viral pharmaceutical composition of the invention to herself,the child and other family members to prevent viral infection andsubsequent illness. People traveling to parts of the world where acertain infectious disease is prevalent (e.g. hepatitis A virus,malaria, etc.) can also be treated.

Alternatively, the pharmaceutical compositions may be used to treattumors or prevent tumor formation, e.g., in patients who have cancer orin those who are at high risk for developing neoplasms or cancer. Forexample, patients with cancer can be treated to prevent furthertumorigenesis. Alternatively, subjects who are or are expected to beexposed to carcinogens can be treated; individuals involved inenvironmental cleanups who may be exposed to pollutants (e.g. asbestos)may be treated. Alternatively, individuals who are to be exposed toradiation can be treated prior to exposure and thereafter (e.g. patientsexposed to high dose radiation or who must take carcinogenic drugs).

The use of the attenuated viruses of the invention as antitumor agentsis based on the Applicants' discovery that an attenuated influenza virusmutant containing a deletion in its IFN-antagonist gene is able toreduce tumor formation in mice. The antitumor properties of theinvention can be at least partially related to their ability to induceIFN and IFN responses. Alternatively, the antitumor properties of theattenuated viruses of the invention can be related to their ability tospecifically grow in and kill tumor cells, many of which are known tohave deficiencies in the IFN system. Regardless of the molecularmechanism(s) responsible for the antitumor properties, the attenuatedviruses of the invention might be used to treat tumors or to preventtumor formation.

The present invention further encompasses the mutant viruses with analtered IFN-antagonist phenotype which are targeted to specific organs,tissues and/or cells in the body in order to induce therapeutic orprophylactic effects locally. One advantage of such an approach is thatthe IFN-inducing viruses of the invention are targeted to specificsites, e.g. the location of a tumor, to induce IFN in a site specificmanner for a therapeutic effect rather than inducing IFN systemicallywhich may have toxic effects.

The mutant IFN-inducing viruses of the invention may be engineered usingthe methods described herein to express proteins or peptides which wouldtarget the viruses to a particular site. In a preferred embodiment, theIFN-inducing viruses would be targeted to sites of tumors. In such anembodiment, the mutant viruses can be engineered to express the antigencombining site of an antibody which recognized the tumor specificantigen, thus targeting the IFN-inducing virus to the tumor. In yetanother embodiment, where the tumor to be targeted expresses a hormonereceptor, such as breast or ovarian tumors which express estrogenreceptors, the IFN-inducing virus may be engineered to express theappropriate hormone. In yet another embodiment, where the tumor to betargeted expresses a receptor to a growth factor, e.g. VEGF, EGF, orPDGF, the IFN-inducing virus may be engineered to express theappropriate growth factor or portion(s) thereof. Thus, in accordancewith the invention, the IFN-inducing viruses may be engineered toexpress any target gene product, including peptides, proteins, such asenzymes, hormones, growth factors, antigens or antibodies, which willfunction to target the virus to a site in need of anti-viral,antibacterial, anti-microbial or anti-cancer activity.

Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The pharmaceutical compositions of thepresent invention may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local. In addition, ina preferred embodiment it may be desirable to introduce thepharmaceutical compositions of the invention into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, N.Y. (1984); Ranger & Peppas, 1983, J. Macromol. Sci.Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;During et al., 1989, Ann. Neurol. 25:351 (1989); Howard et al., 1989, J.Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, i.e., thelung, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, 1984, in Medical Applications of Controlled Release, supra,vol. 2, pp. 115-138). Other controlled release systems are discussed inthe review by Langer (1990, Science 249:1527-1533).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of the attenuated virus, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeiae for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the Therapeutic,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The amount of the pharmaceutical composition of the invention which willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges foradministration are generally about 10⁴-5×10⁶ pfu and can be administeredonce, or multiple times with intervals as often as needed.Pharmaceutical compositions of the present invention comprising10⁴-5×10⁶ pfu of mutant viruses with altered IFN antagonist activity,can be ad ministered intranasally, intratracheally, intramuscularly orsubcutaneously Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

6. EXAMPLE Generation and Characterization of NS1 Truncation Mutants ofInfluenza a Virus 6.1 Material and Methods

Influenza A/PR/8/34 (PR8) virus was propagated in 10-day-old embryonatedchicken eggs at 37° C. Influenza A virus 25A-1, a reassortant viruscontaining the NS segment from the cold-adapted strainA/Leningrad/134/47/57 and the remaining genes from PR8 virus (Egorov etal., 1994, Vopr. Virusol. 39:201-205; Shaw et al., 1996, in Options ofthe control of influenza III, eds. Brown, Hampson Webster (ElsevierScience) pp. 433-436) was grown in Vero cells at 34° C. The 25A-1 virusis temperature sensitive in mammalian cells, and was used as a helpervirus for the rescue of the NS1/99 transfectant virus. Vero cells andMDCK cells maintained in minimal essential medium (MEM) containing 1μg/ml of trypsin (Difco Laboratories, Detroid, Mich.) were used forinfluenza virus growth. Vero cells were also used for selection, plaquepurification and titration of the NS1/99 virus. MDCK cells weremaintained in DMEM (Dulbecco's minimal essential medium) containing 10%heat-inactivated fetal calf serum. Vero cells were grown in AIM-V medium(Life Technologies, Grand Island, N.Y.).

The plasmid pT3NS1/99, which contains a 99 amino acid C-terminaltruncated form of NS1 was made as follows. First, pPUC19-T3/NS PR8,containing the complete NS gene of PR8 virus flanked by the T3 RNApolymerase promoter and BpuAI restriction site was amplified by reversePCR (Ochman et al., 1988, Genetics 120:621-623) using the appropriateprimers. The obtained cDNA thus containing the truncated NS1 gene wasphosphorylated, Klenow treated, self-ligated and propagated in E. colistrain TG1. The construct obtained after purification was namedpT3NS1/99 and verified by sequencing. Plasmids for expression of NP,PB1, PB2, and PA proteins of PR8 virus (pHMG-NP, pHMG-PB1, pHMG-PB2, andpHMG-PA) were previously described (Pleschka et al., 1996, J. Virol.70:4188-4192). pPOLI-NS-RB was made by substituting the CAT open readingframe of pPOLI-CAT-RT (Pleschka et al., 1996, J. Virol. 70:4188-4192)within RT-PCR product derived from the coding region of the NS gene ofinfluenza A/WSN/33 (WSN)virus. This plasmid expresses the NS-specificviral RNA segment of WSN virus under the control of a truncated humanpolymerase I promoter.

Generation of NS1/99 virus was performed by ribonucleoprotein (RNP)transfection (Luytjes et al., 1989, Cell 59:1107-1113). The RNPs wereformed by T3 RNA polymerase transcription from pT3NS1/99 linearized withBpuAI in the presence of purified nucleoprotein and polymerase ofinfluenza 25A-1 virus (Enami, et al., 1991, J. Virol. 65:2711-2713). RNPcomplexes were transfected into Vero cells which were previouslyinfected with 25A-1 virus. Transfected cells were incubated for 18 hoursat 37° C., and the supernatant was passaged twice in Vero cells at 40°C. and plaque purified three times in Vero cells covered with agaroverlay media at 37° C. The isolated NS1/99 virus was analyzed by RT-PCRusing specific primers. The wild-type transfectant virus was generatedas follows: Vero cells in 35-mm dishes were transfected with plasmidspHMG-NP, pHMG-PB1, pHMG-PB2, pHMG-PA and pPOLI-NS-RB, as previouslydescribed (Pleschka et al., 1996, J. Virol. 70:4188-4192). Two dayspost-transfection, cells were infected with 5×10⁴ pfu of delNS1 virusand incubated two more days at 37° C. Cell supernatant was passaged oncein MDCK cells and twice in chicken embryonated eggs. Transfectantviruses were cloned by limiting dilution in eggs. Genomic RNA frompurified NS1/99 transfectant virus was analyzed by polyacrylamide gelelectrophoresis, as previously described (Zheng et al., 1996, Virology217:242-251). Expression of a truncated NS1 protein by NS1/99 virus wasverified by immunoprecipitating labeled infected cell extracts using arabbit polyclonal antisera against NS1.

The allantoic cavity of embryonated chicken eggs, aged 6, 10, and 14days were inoculated with approximate 10³ pfu of PR8, NS1/99, or delNS1(in which the entire NS1 gene is deleted) viruses, incubated at 37° C.for two days, and the viruses present in the allantoic fluid weretitrated by hemagglugination (HA) assay.

Groups of 5 BALB/c mice (Taconic Farms) were inoculated intranasallywith 5×10⁶ pfu, 1.5×10⁵ pfu, or 5×10³ pfu of wild-type A/PR/8/34 (PR8)or NS1/99 virus. Inoculations were performed under anesthesia using 50μl of MEM containing the appropriate number of plaque forming units ofthe appropriate virus. Animals were monitored daily, and sacrificed whenobserved in extremis. In a subsequent experiment, all surviving micewere challenged four weeks later with a dose of 100LD₅₀ of wild-type PR8virus. All procedures were in accord with NIH guidelines on care and useof laboratory animals.

6.2 Results: Attenuation of Influenza A Viruses by NS1 Deletions

Applicants have previously shown that an influenza A virus in which theNS1 gene was deleted (delNS1 virus) is able to grow to titers ofapproximately 10⁷ pfu/ml in cells deficient in type I Interferon (IFN)production, such as Vero cells. However, this virus was impaired in itsability to replicate and cause disease in mice (Garcia-Sastre et al.,1998, Virology 252:324). By contrast, delNS1 virus was able to grow inand kill STAT1−/− mice. These results demonstrated that the NS1 proteinof influenza A virus is a virulence factor involved in the inhibition ofthe host antiviral responses mediated by type I IFN. The followingexperiments were conducted to determine whether one could generateinfluenza viruses with virulence characteristics intermediate betweenwild-type and delNS1 viruses by deleting portions of the NS1 gene andwhether some of these viruses might have optimal characteristics forbeing used as live attenuated vaccines against influenza viruses, i.e.,stability and an appropriate balance between attenuation, immunogenicityand growth in substrates suitable for vaccine preparation, such asembryonated chicken eggs.

In order to test this hypothesis, an influenza A/PR/8/34 (PR8) virus wasgenerated in which the NS1 gene has been modified in order to direct theexpression of a truncated NS1 protein containing only 99 amino acids atthe amino terminal in common with the 230 amino acids of the wild-typeNS1 protein. This virus (NS1-99) was obtained by RNP transfection of anartificially engineered NS gene using 25A-1 helper virus, as previouslydescribed (Garcia-Sastre et al., 1998, Virology 252:324). Analysis ofNS1 expression in virus infected cells revealed the truncated nature ofthe NS1 protein of the NS1-99 virus.

The ability of delNS1, NS1-99 and wild-type PR8 viruses to grow inembryonated chicken eggs of different ages was analyzed. The rationalefor this experiment comes from the fact that the ability of embryonatedeggs to synthesize and to respond to type I IFN under an appropriatestimulus is age dependent. In fact, both IFN inducibility andresponsiveness start at an age of approximately 10 days, and thenexponentially increase with the age (Sekellick et al., 1990, In VitroCell. Dev. Biol. 26:997; Sekellick & Marcus, 1985, J. Interferon Res.5:657). Thus, the use of eggs of different ages represents a uniquesystem to test the ability of different viruses to inhibit IFNresponses. Eggs of 6, 10, and 14 days of age were inoculated withapproximately 10³ pfu of PR8, NS1-99 or delNS1 viruses, incubated at 37°C. for 2 days, and the viruses present in the allantoic fluid weretitrated by hemagglutination (HA) assay. As shown in Table 3, whereaswild-type virus grew to similar HA titers in embryonated eggs of 6, 10and 14 days of age, delNS1 only replicated to a detectable HA titer in6-day-old eggs. By contrast, NS1-99 virus showed an intermediatebehavior between delNS1 and wild-type viruses, and was able to grow toHA titers similar to those of wild-type virus in 10-day-old eggs, butnot in 14-day-old eggs.

TABLE 3 Virus replication in embryonated chicken eggs. Age ofHemagglutination titer¹ Virus eggs: 6 days 10 days 14 days WT PR8² 2,0484,096 1,071 NS1/99 N.D.³ 2,048 <2 de1NS1 64 <2 <2 ¹Titers represent thehighest dilution with hemagglutinating activity. ²Wild-type influenzaA/PR/8/34 virus. ³Not determined.

The attenuation characteristics of NS1-99 virus were next determined inmice. For this purpose, groups of 5 BALB/c mice were intranasallyinfected with 5×10⁶ pfu, 1.5×10⁵ or 1.5×10³ pfu of wild-type PR8 orNS1-99 virus. Mice were then monitored during 3 weeks for survival. Theresults are given in Table 4. NS1-99 virus had an LD50 at least threelogs higher than that of wild-type virus.

TABLE 4 Attenuation of NS1-99 virus in mice Survivors Virus Infectingdose (pfu): 5 × 10⁶ 1.5 × 10⁵ 5 × 10³ WT PR8¹ 1/5 1/5 1/5 NS1-99 3/5 5/55/5 ¹Wild-type Influenza Virus A/PR/8/34.

7. EXAMPLE Generation and characterization of NS1 Truncation Mutants inInfluenza B Virus 7.1 Materials and Methods

Experimental details are similar to those in Section 6.1. Two mutantinfluenza B viruses, B/610B5B/201 B/201) and B/AWBY-234, 127-amino-acidand 90 amino acids in length (C-terminal truncated NS1 proteins),respectively (Norton et al., 1987 Virology 156: 204; Tobita et al., 1990Virology 174:314) were derived from coinfection experiments in tissueculture involving B/Yamagata/1/73 (B/Yam) and A/Aichi/2/68 viruses inthe presence of anti-A (H3N2) virus antibody. The growth of the mutantinfluenza viruses in embryonated eggs of various ages were compared tothat of parental virus B/Yam, which possess a wild-type 281-amino-acidNS1 protein. Eggs of 6, 10 and 14 days of age were inoculated withapproximately 10³ pfu of B/Yam, B/201 or B/AW/BY-234 viruses, incubatedat 35° C. for 2 days, and the viruses present in the allantoic fluidwere titrated by an HA assay.

Further, the attenuation characteristics of B/201 and B/AWBY-234 viruseswere determined in mice. Groups of three BALB/c mice were intranasallyinfected with 3×10⁵ pfu of wild-type B/Yam, B/201 or B/AWBY-234 mutantviruses, and the ability of these viruses to replicate was determined bymeasuring viral titers in lungs at day 3 postinfection since wild-typeB/Yam does not induce apparent signs of disease in mice.

7.2 Results

TABLE 5 Influenza B virus replication in embryonated chicken eggs. Ageof Hemagglutination titer Virus eggs: 6 days 10 days 14 days B/Yam 362256 <2 B/201 32 <2 <2 B/AWBY-234 8 <2 <2

The results from the growth of the mutant and wild-type influenza Bviruses in embryonated chicken eggs, shown in Table 5, demonstrate that,as in the case with influenza A viruses, a carboxy-terminal truncationof the NS1 of influenza B virus is responsible for a lower replicationyield in older embryonated chicken eggs which mount an efficient IFNresponse. This finding indicates that the NS1 of influenza B virus isalso involved in inhibiting the IFN responses of the host, and thatdeletions on the NS1 gene of influenza B virus result in an attenuatedphenotype.

The results from the replication experiments in mice are given in Table6. B/201 and B/AWBY-234 virus titers were approximately three logs ofmagnitude lower that B/Yam titers, indicating that truncations of thecarboxy-terminal domain of the NS1 of influenza B virus are responsiblefor an attenuated phenotype in mice.

TABLE 6 Influenza B virus replication in mouse lungs Virus Lung titersat day 3 postinfection (pfu/lung) B/Yam 2 × 10⁴ 1 × 10⁴ 3 × 10⁴ B/201 30<10 60 B/AWBY-234 <10 40 <10

8. PROTECTION AGAINST WILD-TYPE INFLUENZA VIRUS INFECTION IN MICEIMMUNIZED WITH INFLUENZA A AND B VIRUSES CONTAINING DELETIONS IN THEIRNS1 PROTEINS

In order to determine whether mice immunized with attenuated influenza Aand B viruses containing truncated NS1 proteins were protected againstchallenge with their respective wild-type viruses the followingexperiment was carried out. BALB/c mice were immunized intranasally withA/NS1-99 virus and three weeks later they were infected with 100 LD₅₀ ofwild-type influenza A/PR/8/34 virus. Immunized animals were protectedagainst death, while all control naive mice died after the challenge(see Table 7). In a second experiment, BALB/c mice were intranasallyimmunized with the influenza B viruses B/201 or B/AWBY-234, expressingtruncated NS1 proteins. Three weeks later the mice were challenged with3×10⁵ pfu wild-type influenza B/Yam/1/73 virus. Since this strain ofinfluenza B virus does not induce disease symptoms in mice, the degreeof protection was determined by measuring virus titers in lungs at day 3post-challenge. While naive control animals had titers around 10⁴pfu/lung, viruses were not detected in lungs of immunized animals (seeTable 8). These findings suggest that influenza A as well as influenza Bviruses containing modified NS1 genes are able to induce an immuneresponse in mice which is fully protective against subsequent wild-typevirus challenge.

TABLE 7 Survival of mice immunized with influenza A/NS1-99 virus afterchallenge with 100 LD₅₀ of wild-type influenza A/PR/8/34 virus.Immunizing Dose of A/NS1-99 Virus Number of Survivors/Total   5 × 10⁶pfu 3/3 1.5 × 10⁵ pfu 4/4 PBS 0/5

TABLE 8 Lung titers in mice immunized with influenza B/201 andB/AWBY-234 viruses after challenge with 3 × 10⁵ pfu of wild-typeinfluenza B/Yamagata/73 virus. Immunizing Dose Lung titers (pfu/lung) 3× 10⁵ pfu of B/201 <10¹, <10¹, <10¹, <10¹, <10¹ 3 × 10⁵ pfu ofB/AWBY-234 <10¹, <10¹, <10¹, <10¹, <10¹ PBS 2.5 × 10⁴, 1 × 10⁴, 1.7 ×10⁴, 3 × 10⁴, 5 × 10⁴

9. EXAMPLE Induction of Type I Interferon in Embryonated Eggs Infectedwith DELNS1 Virus

The ability of delNS1 virus, an influenza A virus lacking the NS1 gene,to induce type I IFN secretion in embryonated chicken eggs was nextdetermined. For this purpose, groups of two 10-days-old embryonatedchicken eggs were infected with 5×10³ pfu of delNS1 or wild-type PR8viruses. Eighteen hours postincubation at 37° C., the allantoic fluidwas harvested and dialyzed against acid pH overnight, to inactivateinfectious viruses. After acid pH treatment, samples were dialyzedagainst PBS, and they were tested for IFN activity by determining thehighest dilution with protective activity against VSV infection(approximately 200 pfu) in CEF cells. The results shown in Table 9indicate that in the absence of NS1, influenza A viruses are higherinducers of IFN.

TABLE 9 Induction of IFN in eggs. Virus IFN (U/ml) PR8 <16, <16 de1NS1400, 400 mock <16, <16

10. EXAMPLE Antiviral Activity of delNS1 Virus

Elimination of the IFN antagonist (NS1) gene from influenza A virus mayresult in a virus with the ability to induce high levels of IFN. If thisis the case, delNS1 virus will “interfere” with the replication ofIFN-sensitive viruses. In order to test this possibility, Applicantsinvestigated the ability of delNS1 virus to inhibit the replication ofinfluenza A/WSN/33 (WSN) virus, a commonly used laboratory strain ofinfluenza virus, in eggs. As can be seen in FIG. 1, treatment with only2 pfu of delNS1 virus was able to reduce the final titers of WSN virusin the allantoic fluid by one log. In addition, treatment with 2×10⁴ pfuof delNS1 virus resulted in practically complete abrogation of WSNreplication in eggs. DelNS1 virus was also able to interfere with thereplication in eggs of other influenza A virus strains (H1N1 and H3N2),influenza B virus and a different virus such as Sendai virus (FIG. 2).

Encouraged by these results, Applicants next determined the ability ofdelNS1 virus to interfere with wild-type influenza virus replication inmice. Although type I IFN treatment in tissue culture prevents influenzaA virus replication in vitro, treatment of mice with IFN is not able toinhibit the replication of influenza viruses (Haller, 1981, Current TopMicrobiol Immunol 92:25-52). This is true for most inbred strains ofmice, except for A2G mice. A2G mice, as well as a significant proportionof wild mice (approximately 75%), contain at least one intact Mx1allele, while most laboratory strains are Mx1−/− (Haller, 1986, CurrentTop Microbiol Immunol 127:331-337). The Mx1 protein, which is ahomologue of the human MxA protein (Aebi, 1989, Mol. Cell. Biol.11:5062), is a potent inhibitor of influenza virus replication (Haller,1980, Nature 283:660). This protein is not constitutively expressed, butits expression is transcriptionally induced by type I IFN. Thus, A2Gmice can be used to test the ability of IFN-inducers to stimulate anantiviral response against influenza A viruses (Haller, 1981, CurrentTop Microbiol Immunol 92:25-52).

Applicants intranasally infected eight 4-week-old A2G mice with 5×10⁶pfu of a highly pathogenic influenza A/PR/8/34 virus isolate (Haller,1981, Current Top Microbiol Immunol 92:25-52). Half of the mice receivedan intranasal treatment with 5×10⁶ pfu of delNS1 at −24 h with respectto the PR8 infection. The other four mice were treated with PBS. Bodyweight changes and survival was monitored. These results demonstratethat delNS1 treatment was able to protect A2G mice against influenzavirus-induced death and body weight lost. The same treatment was noteffective in Mx1−/−mice indicating that the mechanism of viralprotection was Mx1, i.e. IFN, mediated.

11. EXAMPLE Antitumor Properties of DELNS1 Virus in Mice

Given that type I IFN and/or inducers of type I IFN have been shown tohave antitumor activities (Belardelli and Gresser, 1996 Immunology Today17: 369-372; Qin et al., 1998, Proc. Natl. Acad. Sci. 95: 14411-14416),it is possible that treatment of tumors with delNS1 virus might mediatetumor regression. Alternatively, delNS1 virus might have oncolyticproperties, i.e., it may be able to specifically grow in and kill tumorcells, many of which are known to have deficiencies in the IFN system.In order to test anti-tumor activity of delNS1 virus, the followingexperiment was conducted using murine carcinoma cell line CT26.WT in amouse tumor model for pulmonary metastasis (Restifo et al., 1998Virology 249:89-97). 5×10⁵ CT26.WT cells were injected intravenouslyinto twelve 6-weeks-old BALB/c mice. Half of the mice were treatedintranasally with 10⁶ pfu of delNS1 virus every 24 hours at days 1, 2and 3 postinoculation.

Twelve days after tumor injection, mice were sacrificed and lungmetastases were enumerated. As shown in Table 10, delNS1 treatmentmediated a significant regression of murine pulmonary metastases.

TABLE 10 Antitumor activity of de1NS1 virus in BALB/C mice injected withCT26.WT tumor cells. Number of pulmonary metastases PBS-treatedde1NS1-treated Mouse 1 >250 120 Mouse 2 >250 28 Mouse 3 >250 9 Mouse4 >250 6 Mouse 5 >250 2 Mouse 6 >250 1

12. EXAMPLE The NS1 Protein Inhibits the Translocation of IRF-3 DuringInfluenza Virus Infection

The results described herein suggest that the NS1 protein of influenzavirus is responsible for the inhibition of the type I IFN responseagainst the virus, and that mutations/deletions in this protein resultin attenuated viruses due to an enhanced IFN response during infection.It is known that synthesis of type I IFN during viral infection can betriggered by double-stranded RNA (dsRNA). IRF-3 is a transcriptionfactor which is usually found in an inactive form in the cytoplasm ofmammalian cells. Double-stranded RNA induces the phosphorylation(activation) of the transcription factor IRF-3, resulting in itstranslocation to the nucleus, where it induces transcription of specificgenes, including genes coding for type I IFN (Weaver et al., 1998, Mol.Cell. Biol. 18:1359). In order to determine if NS1 of influenza isacting on IRF-3, IRF-3 localization in CV1 cells infected with wild-typePR8 or with delNS1 influenza A virus was monitored. FIG. 3 shows thatIRF-3 translocation is minimal in PR8-infected cells (in fewer than 10%of the cells). In contrast, approximately 90% of delNS1-infected cellsshowed nuclear localization of IRF-3. Strikingly, it was possible topartially inhibit the IRF-3 translocation in delNS1-infected cells byexpressing NS1 from a plasmid in trans. The results demonstrate that theNS1 of influenza A virus is able to inhibit IRF-3 translocation invirus-infected cells. It is likely that the NS1 of influenza virusprevents dsRNA-mediated activation of IRF-3 by sequestering the dsRNAgenerated during viral infection, thus resulting in an inhibition of IFNsynthesis.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any constructs or viruses whichare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed:
 1. A method for vaccine production comprising: (a)propagating in an interferon deficient substrate an attenuated influenzavirus having a mutation in the NS1 gene that diminishes or eliminatesthe ability of the NS1 gene product to antagonize the cellularinterferon response; and (b) collecting progeny virus, wherein theinterferon deficient substrate is the allantoic cavity of an immatureembryonated egg six to nine days old or an interferon deficient cellline, with the proviso that said virus is not influenza C virus and theinterferon deficient substrate is not Vero cells or STAT1(−) cell lines.2. A method for vaccine production comprising: (a) propagating inembryonated eggs less than ten days old a recombinantly engineeredattenuated influenza virus having a mutation in the NS1 gene thatdiminishes or eliminates the ability of the NS1 gene product toantagonize the cellular interferon response; and (b) collecting progenyvirus, wherein the virus is grown to sufficient quantities and underconditions that are free from contamination, such that the progeny virusis suitable for formulation into a vaccine, with the proviso that saidvirus is not influenza C virus.
 3. The method of claim 1, wherein theattenuated influenza virus is genetically engineered.
 4. The method ofclaim 1 or 2, wherein the mutation in the NS1 gene is a deletion at theC-terminal of NS1.
 5. The method of claim 4, wherein the NS1 geneencodes truncated NS1 proteins consisting of amino acid residues 1-60,amino acid residues 1-70, amino acid residues 1-90, amino acid residues1-99, amino acid residues 1-100, amino acid residues 1-110, amino acidresidues 1-120, amino acid residues 1-124, or amino acid residues 1-130of the wild-type NS1.
 6. The method of claim 2, wherein the egg is a sixto nine days old chick egg.
 7. The method of claim 1, wherein theinterferon deficient substrate is the allantoic cavity of an immatureembryonated egg six days old.
 8. The method of claim 1 or 2, wherein theinfluenza virus is influenza A or B.
 9. The method of claim 1 or 2,wherein the mutation in the NS1 gene is responsible for the attenuatedphenotype of the influenza virus.
 10. The method of claim 2, wherein thevirus is propagated in the allantoic cavity of embryonated chick eggs.11. A method for vaccine production comprising: (a) propagatinginfluenza virus in the allantoic cavity of an immature embryonated chickegg six days old; and (b) collecting progeny virus, wherein the virus isgrown to sufficient quantities and under conditions that are free fromcontamination, such that the progeny virus is suitable for formulationinto a vaccine, wherein the influenza virus is influenza A or B.